HPV16重组伪病毒靶向Gd-B细胞递送miR-148b海绵基因纠正IgA1糖基化缺陷的体内外研究

Underglycosylated or Gal-deficient IgA1 (Gd-IgA1) plays a key role in the pathogenesis of IgA nephropathy (IgAN). Aberrantly IgA1 glycosylation is partly due to the low activities of galactosyltransferase C1GalT1, which is a target gene negatively regulated by miR-148b. Over expression of miR-148b promotes Gd-IgA1 production has been observed in patients with IgAN. In this project, we aimed to prevent miR-148b mediated C1GalT1 gene silencing by delivering a miRNA sponge tareging miR-148b to Gd-IgA1-producing B cells. We first insert a DNA segment encoding peptide PH, which mimics the HAA associating with Gd-IgA1, into the 798/799 site of capsid L1 gene of human papillary virus type 16 (HPV 16) by PCR site-specific mutation. The chimerical L1-PH VLP are produced and used to package the plasmid encoding miR-148b sponge, come to be the pseudovirus L1-PH/pmiR-148b-SP. The PH peptide outside the surface of this pseudovirus will direct the recognizing and engagement with membrane Gd-IgA1 in Gd-B cells and further being internalized. The internalized pmiR-148b-SP will be expressed and elicit the function of down-regulating miR-148b, thus reduce miR-148b mediated C1GalT1 mRNA silencing. The effects of correcting IgA1 underglycosylation in Gd-B cell will be evaluated in vitro and in vivo. We hope our project will shed light on IgAN therapy by combined application of miRNA strategy and pseudovirus-based drug delivering.

分泌IgA1的B细胞（Gd-B）异常高表达miR-148b，使半乳糖基转移酶C1GalT1合成不足，产生半乳糖基缺陷的IgA1（Gd-IgA1），Gd-IgA1刺激机体产生抗体，形成免疫复合物导致IgA肾病发生。本项目拟联合应用伪病毒载体技术和miRNA海绵策略，靶向性修正Gd-B细胞内IgA1糖基化异常。首先将编码HAA模拟肽PH（与Gd-IgA1具亲和性）基因定点插入HPV16衣壳蛋白L1（HPV16L1），表达制备L1-PH嵌合病毒样颗粒（VLP）；另构建编码靶向miR-148b的miRNA海绵表达载体pmiR-148b-SP。利用L1-PH VLP自包装能力，将二者包装成L1-PH／miR-148b-SP HPV16伪病毒。通过体内外实验观察新型伪病毒特异性感染Gd-B细胞、抑制miR-148b介导的C1GalT1基因消减、纠正IgA1糖基化缺陷以及对IgA肾病的治疗作用。

分泌IgA1的B细胞（Gd-B）异常高表达miR-148b，使半乳糖基转移酶C1GalT1合成不足，产生半乳糖基缺陷的IgA1（Gd-IgA1），Gd-IgA1刺激机体产生抗体，形成免疫复合物导致IgA肾病发生。本项目联合应用伪病毒载体技术和miRNA海绵策略，靶向性修正Gd-B细胞内IgA1糖基化异常。首先将编码HAA模拟肽PH（与Gd-IgA1具亲和性）基因定点插入HPV16衣壳蛋白L1（HPV16L1），表达制备L1-PH嵌合病毒样颗粒（VLP）；另构建编码靶向miR-148b的miRNA海绵表达载体pmiR-148b-SP。利用L1-PH VLP自包装能力，将二者包装成L1-PH／miR-148b-SP HPV16伪病毒。我们发现L1-PH／miR-148b-SP HPV16新型伪病毒能特异性感染Gd-B细胞，并将miR-148b-SP HPV16海绵基因导入细胞内并表达，此海绵基因能抑制miR-148b介导的C1GalT1基因消减，从而纠正IgA1糖基化缺陷，减少Gd-B细胞合成Gd-IgA1的水平，从而对IgA产生治疗作用。本研究探索了一种新的针对IgA肾病的精准治疗策略。